Arachidonic acid (AA) alone or via its metabolism through the cyclooxygenase (COX), lipooxygenase (LOX) and cytochrome P450 monooxygease (CYP) pathways plays an important role in the regulation of cell signaling events that are either crucial in the maintenance of homeostasis or disease pathogenesis. Phospholipase A2 (PLA2), phospholipase D (PLD) and phospholipase C (PLC) mediate AA release in response to stimulus. Among these three phospholipases, PLA2 plays a rate- limiting role in stimulus-induced AA release. During the previous period of the present grant proposal, we demonstrated a potent role for the LOX metabolites of AA in the regulation of angiogenesis using microvascular endothelial cells from different vascular beds, including retinal vascular bed. While angiogenesis is essential for the development and wound healing, it also plays a progressive role in various disease processes including cancer and diabetic retinopathy. A large body of data showed that angiogenic growth factors, particularly vascular endothelial growth factor (VEGF) plays a determinant role in the pathogenesis of diabetic retinopathy. Although the signaling mechanisms underlying VEGF-induced angiogenesis are fairly well studied, the involvement of AA in its angiogenic signaling events is not explored. Towards filling this gap, we accidentally discovered that VEGF- induced AA release depends on PLD activation in human retinal microvascular endothelial cells (HRMVEC). Since inhibition of PLA2 but not diacylglycerol (DAG) lipase suppressed VEGF-induced AA release, it is likely that VEGF-induced AA release is mediated by PLD-dependent PLA2 activation. Surprisingly, inhibition of PLD or PLA2 also attenuated VEGF-induced HRMVEC DNA synthesis, migration and tube formation. Based on these novel findings, we hypothesize that PLD-dependent PLA2 activation plays a determinant role in VEGF-induced retinal angiogenesis. To test this hypothesis, we will propose and address the following three specific aims. Aim 1. To test the hypothesis that PLD mediates VEGF-induced angiogenesis. Aim 2. To test the hypothesis that PLD- dependent PLA2 activation is required for VEGF-induced angiogenesis. Aim 3. To test the hypothesis that PLD-dependent PLA2 activation plays a determinant role in retinal angiogenesis. The results of the experiments proposed in the above-listed three specific aims will provide novel information in regard to the role of PLD-PLA2 axis in the regulation of retinal angiogenesis and thereby in retinal diseases. PUBLIC HEALTH RELEVANCE: Angiogenesis plays a major role in retinal diseases such as diabetic retinopathy. Understanding the mechanisms underlying retinal angiogenesis is, therefore, crucial in the development of therapeutic agents against these ocular lesions. The present research proposal seeks to study the role of PLD- PLA2 axis in VEGF-induced retinal angiogenesis and thereby in retinopathies.